ИСТИНА

The highly selective binding of a ligand to a targeted protein is a crucial event in the molecular mechanism of action for many drugs. For example, selective inhibition of a bacterial versus a related human enzyme is a key factor determining the efficiency and low toxicity of an antibacterial drug. Understanding the factors that determine such selectivity is of practical as well as fundamental theoretical interest. An important model for such studies is the enzyme dihydrofolate reductase (DHFR) which is the target for a number of clinically useful antibacterial and antineoplastic drugs. One such drug, trimethoprim (TMP), binds to the bacterial enzyme about 3,000 times tighter than it does to the mammalian form. Most of this increase comes from the high co-operative binding effect between TMP and the NADPH co-enzyme when these ligands bind to the bacterial enzyme. It is clearly important to understand the molecular basis for such co-operativity and specificity of binding. We have used a combination of NMR spectroscopy and various computational techniques to attempt to understand some of the factors that control specificity of trimethoprim binding to bacterial (Lactobacillus casei) DHFR. High resolution solution structures of both the binary DHFR-TMP and ternary DHFR-TMP-NADPH complexes have been determined. Quantum mechanical calculations have been used to study experimentally determined specific interactions between ligands and DHFR residues. In order to obtain comprehensive information about the trimethoprim - DHFR interactions, static structural data from the NMR solution structure determinations have been complemented by information on molecular motions in the DHFR complexes and specific interactions directly monitored within the binding site. Molecular motions have been studied using both NMR relaxation measurements and molecular dynamic simulations of DHFR complexes.